The relentless rise in multidrug resistance exerts devastating human and economic tolls, heralding a public health crisis. A major mechanism underlying multidrug resistance is mediated by integral membrane proteins called multidrug transporters, which can act as promiscuous molecular pumps to flush therapeutic drugs out of cells. Multidrug and toxic compound extrusion (MATE) proteins constitute a ubiquitous family of multidrug transporters and couple the efflux of structurally dissimilar, typically cationic compounds to the influx of Na+ or H+. The ~900 MATE transporters identified to date can be separated into the NorM, DinF (DNA-damage-inducible protein F) and eukaryotic subfamilies based on amino-acid sequence similarity. MATE transporters hold great appeal as novel therapeutic targets as they can extrude a litany of antibiotics, anticancer and diabetic drugs across cell membranes. The X-ray structures of several Na+-coupled NorM and H+-coupled DinF transporters, all captured in the extracellular-facing states, have been reported by us and others. Our substrate-bound structures in particular suggested how NorM and DinF transporters recognize and extrude cationic drugs via different mechanisms. Despite such triumphs, little is known about how a MATE transporter selects the counter-transported cation, or how it alternates between the extracellular- and intracellular-facing conformations to expel drugs. To fil such glaring gaps in our knowledge, we will elucidate the molecular structures of MATE transporters at conformation states distinct from the known structures. Based on those structures, we will deduce testable hypotheses, construct MATE mutants, and examine their transport function. Our long-term goal is to acquire a detailed, mechanistic understanding of multidrug transport. The current study will accomplish the following aims: (1) to determine the X-ray structures of H+- and Na+-coupled DinF transporters in their cation-bound states; (2) to visualize intracellular-facing MATE transporters by combining cross-linking with biochemical and crystallographic methods; (3) to enable mechanistic interpretation of our structures by conducting drug accumulation, antiport, efflux and resistance assays. Our findings will shine new light on the molecular underpinnings of multidrug resistance and open new vistas on therapeutic intervention.